Diatoms are a major phylum of the phytoplankton biodiversity in oceans, fresh water and various soil habitats. They are responsible for up to 25% of the global primary productivity. Study of this group of eukaryotes has benefited from recent developments on two species, Phaeodactylum tricornutum, a model of pennate diatoms and Thalassiosira pseudonana, a model of marine centric diatoms, for which intense efforts have been put to sequence their genomes, define reference data for transcriptome and whole-cell proteome studies, and eventually develop molecular tools to speed up functional analyses. Molecular tools thus allow the deciphering of the role of proteins by gene knockdown technologies (RNAi) and functional characterization of obtained genetically engineered lines. Availability of models for both pennate and centric diatoms is also essential to address common or specific features of these two groups.
Diatoms, like other microalgae, are considered a plausible alternative source of hydrocarbons to replace fossil fuels, with the advantage of having a neutral CO2 balance, based on the hypotheses that CO2 and water can be efficiently converted into biomass by photosynthesis and that the carbon metabolism could be controlled so that they accumulate energetically-rich triacylglycerol (TAG, also called oil). Different phytoplanktonic organisms of the Chromalevolata superphylum have focused the attention for their ability to accumulate TAG, with promising initial yields and appropriate robustness and physical properties to be implemented in an industrial process, including P. tricornutum. P. tricornutum is currently used for the industrial production of omega-3 polyunsaturated fatty acids but industrial implementation for biofuels is still limited by the growth retardation and low yield in biomass when TAG accumulation is triggered using conventional nutrient starvation approaches, such as nitrogen starvation. P. tricornutum exhibits interesting properties for an industrial implementation, like the ability to grow in the absence of silicon or the sedimentation of cells that could be useful for harvesting techniques. Progresses in genetic engineering and farming performances are therefore needed for biofuel applications. Attempts to promote TAG accumulation can rely on various strategies that can be combined, including the stimulation of fatty acid and TAG biosynthesis, the blocking of pathways that divert carbon to alternative metabolic routes and eventually the arrest of TAG catabolism.
Many proteins associate with oil droplets and control the storage or release of TAG used for energy, membrane biogenesis or signaling. The CGI-58 (comparative gene identification 58), an α/β hydrolase-type protein primarily, is one of these lipid droplet proteins involved in TAG hydrolysis in mammals. In mice, CGI-58 knockdown thus induced a 4-fold increase in hepatic cells. A CGI-58 homolog exists in angiosperms and its knock out in Arabidopsis led to the accumulation of oil droplets in leaf tissues, which normally do not store lipids. In mammals, the activity of CGI-58 is regulated by a protein called perilipin. No homologs of this protein have been identified in angiosperms. In Arabidopsis, CGI-58 was shown to interact with PXA1, a fatty acid transporter at the surface of peroxisomes, feeding peroxisomal β-oxidation catabolic route. Thus, CGI-58 appears as a protein localized at the surface of oil droplets and interacting with various protein partners from animals to plants, involved in TAG catabolism. The action of CGI-58 occurs therefore by different mechanisms in different organisms, with distinct protein partners following the species (e.g. perilipin in mammals or PXA1 in angiosperms). Potential protein partners in diatoms have not been demonstrated.
Description
The present invention relates to a modified protist strain in which the activity of the protein CGI-58 or one of its homologous has been modified in order to permit the accumulation of oil, advantageously of triacylglycerol.
In the present text modified means that the algae strain has been manipulated in order to activate or decrease, eventually until the complete inhibition, the CGI-58 protein activity. Preferably according to the invention the activity of the CGI-58 protein is decrease or completely inhibited.
Many technics are known to alter the expression of a protein. It is possible to cite the technics that alter the gene coding the protein or its expression as for example by mutation, insertion, deletion, RNAi inhibition. These types of technics can be grouped under the term “genetically engineered”.
It is also possible to cite technics that alter the transcription of the gene or the translation of the RNA issued from the transcription of the gene.
It is also possible to cite technics that use at least a compound like a chemical compound or a biological compound (antibody for example) that will alter the activity of the protein for example by binding to it.
According to the invention all known methods that permit to modify the activity of the CGI-58 protein can be used.
According to the invention one of the preferred methods is to genetically modify the expression of the CGI-58 gene or homologous gene thereof in order to at least attenuate its expression, preferably to silence it.
Preferably, the protist organism or protist strain belongs to the kingdom Chromalveolata.
The present invention thus relates to a modified strain of a species belonging to the kingdom Chromalveolata, in which the CGI-58 protein or one of its homologous has been modified in order to permit the accumulation of oil in the strain, advantageously accumulation of triacylglycerol. According to the invention the activity of said protein is impaired, ie at least reduced, preferentially abolished.
In one embodiment of the invention the expression of the CGI-58 gene or any homologous gene thereof is attenuated or silenced (for example, by knocking down).
If the gene is silenced, there is no gene expression and CGI-58 protein synthesis.
If the gene is attenuated, the expression of the gene and the synthesis of CGI-58 protein are decreased of at least 50%, preferably of at least 70% and more preferably at least 90%.
More preferably, the modified strain is respectively a diatom or diatom strain, still more preferably a pennate diatom or a pennate diatom strain.
Advantageously, the pennate diatom or pennate diatom strain is of the Phaeodactylum genus, and more preferably of the strain Phaeodactylum tricornutum. 
An example of Phaeodactylum tricornutum strain is Phaeodactylum tricornutum (Pt1) Bohlin Strain 8.6 CCMP2561 (Culture Collection of Marine Phytoplankton, now known as NCMA: National Center for Marine Algae and Microbiota).
“Homologuous sequence” as used herein refers to a sequence involved in triacylglycerol (TAG) catabolism and having similarity or identity with CGI-58 sequence, with identity being preferred. Homology can be determined using standard techniques known in the art.
In particular, by “% identity” with respect to the homo sapiens CGI-58 sequence is defined herein as the percentage of amino acid residue in a candidate sequence that are identical with the amino acid residues in the CGI-58 sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. The % identity values used herein are generated by EMBOSS (6.3.1) (The European Molecular Biology Open Software Suite (2000) Rice, P. Longden, I. and Bleasby, A. Trends in Genetics 16, (6) pp 276-277).
By “% similarity” with respect to the homo sapiens CGI-58 sequence is defined herein as the percentage of amino acid residues in a candidate sequence that are conserved compared to the amino acid residues in the homo sapiens CGI-58 sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence similarity. So the “% similarity” is the “% identity” plus the percentage of conserved substitution (ie: aspartate for glutamate). The % similarity values used herein are generated by EMBOSS (6.3.1).
Preferably, the homologous gene of the CGI-58 presents at least 15% similarity, at least 20% similarity, more preferably at least 25% similarity and still more preferably at least 30% similarity with the Homo sapiens CGI-58 sequence.
Preferably, the homologous gene of the CGI-58 presents at least 15% identity, and more preferably, at least 20% identity with the Homo sapiens CGI-58 sequence.
As an example, the following data, relating to the Homo sapiens CGI-58 gene and different homologuous genes thereof, can be given:                Homo sapiens (CAD12731)/Arabidopsis thaliana (ABM06019): identity 26.8%, similarities 41.5°%        Homo sapiens (CAD12731)/Phaeodactylum tricornutum (XP_002183583): identity 21.6%, similarities 32.2%        Homo sapiens (CAD12731)/Thalassiosira pseudonana (XP_002294083) non complete sequence: identity 19.3%, similarities 32.1%        Arabidopsis thaliana (ABM06019)/Phaeodactylum tricornutum (XP_002183583): identity 26%, similarities 42.6%        Arabidopsis thaliana (ABM06019)/Thalassiosira pseudonana (XP_002294083) non Complete sequence: identity 20%, similarities 29.1%        Phaeodactylum tricornutum (XP_002183583)/Thalassiosira pseudonana (XP_002294083) non complete sequence: identity 30.9%, similarities 41.2%        
Alternatively, the CGI-58 sequence of reference for the evaluation of the similarity can be the Phaeodactylum tricornutum sequence mentioned above.
The modified strain according to the invention can accumulate or contain at least 1.5 fold, preferably 4 fold, the triacylglycerol content of the corresponding wild type strain.
By “corresponding wild type strain”, it is meant the strain, before the modification aiming at silencing or attenuating the CGI-58 protein activity, preferably the expression of the CGI-58 gene or any homologous gene thereof (i.e. untransformed organism or strain).
Indeed, the inventors have shown that protists harboring the silencing construction contain more oil (>than 1.5 fold increase). In particular, the invention allows to reach a 4 fold increase. Additionally, they have also shown that:                Protists harboring the silencing construction contain more oil in growing medium (such as ESAVV) containing nitrogen (also called “nitrogen enriched medium”, such as Sodium Nitrate, NaNO3 0.05 g/L or 0.034 g/L of N element) or depleted of nitrogen (no addition) than wild type untransformed cells;        Protists harboring the silencing construction contain more oil than wild type untransformed cells;        Protists harboring the silencing construction accumulate oil earlier than wild type untransformed cells;        The accumulation of oil occurs in the early logarithm phase of growth;        The accumulation of oil does not correlate with a retardation of growth.        
The present invention further discloses a method of preparation of a genetically engineered organism or strain according to the invention, comprising the transformation of an organism with a vector expressing RNAi construction designed to target the expression of the CGI-58 gene or any homologous gene thereof.
Advantageously, the vector is introduced in the organism by biolistic methods (particle bombardment) or electroporation.
After transformation, organisms in which the expression of the CGI-58 gene or any homologous gene thereof is attenuated or silenced, are selected and cultured.
The present invention further discloses a method of accumulation of triacylglycerol in an organism belonging to the kingdom Chromalveolata, comprising the step of silencing the expression of the CGI-58 gene or any homologous gene thereof in said organism.
Advantageously, in said method of accumulation, the organism is cultured in an nitrogen-containing medium or alternatively in a nitrogen-depleted medium.
After 1 day (preferably 3 days) in the culture medium, the genetically engineering organisms are harvested and triacylglycerols are recovered.
The invention further encompasses the use of a genetically engineered organism or strain according to the invention for the production of triacylglycerol(s).